Project description:BACKGROUND: Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. RESULTS: We have identified the trxB1-encoded thioredoxin reductase (TR) as a key enzyme in the oxidative stress response of Lactobacillus plantarum WCFS1.Overexpression of the trxB1 gene resulted in a 3-fold higher TR activity in comparison to the wild-type strain. Subsequently, higher TR activity was associated with an increased resistance towards oxidative stress. We further determined the global transcriptional response to hydrogen peroxide stress in the trxB1-overexpression and wild-type strains grown in continuous cultures. Hydrogen peroxide stress and overproduction of TR collectively resulted in the up-regulation of 267 genes. Additionally, gene expression profiling showed significant differential expression of 27 genes in the trxB1-overexpression strain. Over expression of trxB1 was found to activate genes associated with DNA repair and stress mechanisms as well as genes associated with the activity of biosynthetic pathways for purine and sulfur-containing amino acids. A total of 16 genes showed a response to both TR overproduction and hydrogen peroxide stress. These genes are involved in the purine metabolism, energy metabolism (gapB) as well as in stress-response (groEL, npr2), and manganese transport (mntH2). CONCLUSION: Based on our findings we propose that overproduction of the trxB1-encoded TR in L. plantarum improves tolerance towards oxidative stress. This response coincides with simultaneous induction of a group of 16 transcripts of genes. Within this group of genes, most are associated with oxidative stress response. The obtained crossover between datasets may explain the phenotype of the trxB1-overexpression strain, which appears to be prepared for encountering oxidative stress. This latter property can be used for engineering robustness towards oxidative stress in industrial strains of L. plantarum.
Project description:Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.
Project description:There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.
Project description:Dietary fats may exert selective pressures on Lactobacillus species, however, knowledge on the mechanisms of adaptation to fat stress in these organisms is still fragmentary. This study was undertaken to gain insight into the mechanisms of adaptation of Lactobacillus plantarum WCFS1 to olive oil challenge by whole genome transcriptional profiling using DNA microarrays. A set of 230 genes were differentially expressed by L. plantarum WCFS1 to respond to this vegetable oil. This response involved elements typical of the stringent response, as indicated by the induction of genes involved in stress-related pathways and downregulation of genes related to processes associated with rapid growth. A set of genes involved in the transport and metabolism of compatible solutes were downregulated, indicating that this organism does not require osmoprotective mechanisms in presence of olive oil. The fatty acid biosynthetic pathway was thoroughly downregulated at the transcriptional level, which coincided with a diminished expression of genes controlled by this pathway in other organisms and that are required for the respiratory function, pyruvate dehydrogenase activity, RNA processing and cell size setting. Finally, a set of genes involved in host-cell signaling by L. plantarum were differentially regulated indicating that olive oil can influence the expression of metabolic traits involved in the crosstalk between this bacterium and the host.
Project description:There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as potential protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against WCFS1 (pTRKH2) as the indicator strain, showing that functional expression of plantacyclin B21AG had been achieved. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 results in similar killing activity being observed in both the wild type and recombinant hosts despite the lower copy number of pCycB21.
Project description:Lactobacillus species are a predominant member of the vaginal microflora and are critical in maintaining an acidic vaginal environment thought to contribute to the prevention of a number of urogenital diseases. However, during menstruation the pH of the vaginal environment increases to neutrality, a pH conducive for Staphylococcus aureus proliferation and the production of toxic shock syndrome toxin 1 (TSST-1) in susceptible women. In order to generate Lactobacillus species capable of expressing lysostaphin (an endopeptidase that cleaves the cell wall of S. aureus) in a modified genital tract secretion medium (mGTS) under neutral-pH conditions, six prominent proteins from Lactobacillus plantarum WCFS1 spent medium were identified by mass spectrometry. Sequences for promoters, signal peptides, and mature lysostaphin were used to construct plasmids that were subsequently transformed into L. plantarum WCFS1. The promoter and signal sequences of Lp_3014 (putatively identified as a transglycosylase) or the promoter sequence of Lp_0789 (putatively identified as glyceraldehyde 3-phosphate dehydrogenase) with the signal sequence of Lp_3014 exhibited lysostaphin activity on buffered medium containing heat-killed S. aureus. The cassettes were integrated into the chromosome of L. plantarum WCFS1, but only the cassette containing the promoter and signal sequence from Lp_3014 had integrated into the appropriate site. Coculture assays using buffered mGTS showed that lysostaphin expressed from L. plantarum WCFS1 reduced the growth of TSST-1-producing strains of S. aureus under neutral-pH conditions. This study provides the basis for determining whether lysostaphin-producing Lactobacillus strains could potentially be used as a means to inhibit the growth of S. aureus during menstruation.
Project description:BACKGROUND: Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells. RESULTS: Metabolite formation and gene expression were determined in a folate-overproducing- and wild-type strain. Differential metabolomics analysis of intracellular metabolite pools indicated that the pool sizes of 18 metabolites differed significantly between these strains. The gene expression profile was determined for both strains in pH-regulated chemostat culture and batch culture. Apart from the expected overexpression of the 6 genes of the folate gene cluster, no other genes were found to be differentially expressed both in continuous and batch cultures. The discrepancy between the low transcriptome and metabolome response and the 25% growth rate reduction of the folate overproducing strain was further investigated. Folate production per se could be ruled out as a contributing factor, since in the absence of folate production the growth rate of the overproducer was also reduced by 25%. The higher metabolic costs for DNA and RNA biosynthesis in the folate overproducing strain were also ruled out. However, it was demonstrated that folate-specific mRNAs and proteins constitute 8% and 4% of the total mRNA and protein pool, respectively. CONCLUSION: Folate overproduction leads to very little change in metabolite levels or overall transcript profile, while at the same time the growth rate is reduced drastically. This shows that Lactobacillus plantarum WCFS1 is unable to respond to this growth rate reduction, most likely because the growth-related transcripts and proteins are diluted by the enormous amount of gratuitous folate-related transcripts and proteins.
Project description:Whole genome transcriptional profiling was used to characterize the response of Lactobacillus plantarum WCFS1 human isolate during challenge with oleuropein. Twelve independent experiments were performed and mixed at random in groups of four for total of three RNA samples. The transcriptional profile shows that Lactobacillus plantarum WCFS1 adapts its metabolic capacity to acquire certain carbohydrates and repress the expression of genes involved in fatty acid biosyntheis. The transcriptomic datasets also revealed the downregulation of genes related to the biosynthesis of capsular polysaccharides and genes coding for ABC-type transporters. In addition, induction of oligopeptide permeases is also part of the response of Lactobacillus plantarum WCFS1 to oleuropein. Overall design: Biological triplicates: untreated cells: Control vs. Oleuropein treated cells: Experimental
Project description:In order to understand LBG derived galacto-manno-oligosaccharides utilization by a probiotic bacterium, Lactobacillus plantarum WCFS1, we have grown Lactobacillus plantarum WCFS1 (in duplicates) till mid log phase (OD600nm ~0.5, 10 h) in carbon free MRS (de Man, Rogosa Sharpe ) media containing either galacto-manno-oligosaccharides, mannose, glucose or galactose (1% w/v) as the sole carbon source. Overall design: Organism : Lactobacillus plantarum, Agilent's lactobacillus Gene Expression 8x15k Array (AMADID: 067475) designed by Genotypic Technology private Limited